(1) Field of the Invention
The invention relates to laser scanning microscopes, in general, and to laser scanning microscopes that use different excitations wavelengths for different dyes, in particular.
(2) Description of Related Art Including Information Disclosed UNDER 37 CFR 1.97 AND 1.98
A laser scanning microscope (LSM) can be divided essentially in four modules as shown in FIG. 1: light source, scan module, detection unit and microscope. These modules are described in more detail in the DE19702753A1 and U.S. Pat. No. 6,563,632 B1 which are incorporated by reference herein as if reproduced in full.
Lasers with different wavelengths are used in the LSM for specific excitation of the different color substances or dyes in a preparation. The selection of the excitation wavelength is based on the absorption properties of the dye to be tested. The excitation light is created in the light sources module LS. For this different lasers are used here (Argon, Argon Krypton, TiSa-Laser). The combining of different wavelengths in the light source module is done through beam splitter BS or mirror as shown in FIG. 1 in the light source module LS. Further the selection of wavelengths and the setting of intensity of the required excitation wavelength is also done in the light source module, e.g., through the use of an attenuator AT such as an acoustic-optical crystal (AOTF). Subsequently, the illumination ray from the laser reaches the scan module through a fiber FB or a suitable mirror arrangement.
The illumination ray created in the light source is focused on the preparation with the help of the lens diffraction limited through the scanner SC, the scan optics SO and the tubular lens TL. The focus scans the specimen SP in point raster in the x-y direction. The pixel dwell times when scanning over the specimen lie mostly in the range of less than a microsecond up to few seconds.
In confocal detection (descanned detection) of the fluorescence light, the detection light, which is emitted from the focal plane of the specimen SP and from the planes above and below it, reaches a dichroic beam splitter (MDB) via the scanner. This separates the fluorescence light from the excitation light. Subsequently, the fluorescence light is focused on a slit (confocal slit/pinhole), which is exactly in a plane conjugated to the focal plane. Through this the fluorescence light components which are outside the focus are suppressed. Through varying of the size of the pinhole, the optical resolution of the microscope can be set.
Behind the pinhole PH is a further dichroic block filter EF which suppresses the excitation light once again. The fluorescence light is measured by means of a point detector PMT after passing through the block filter EF.
With the use of multiphoton absorption, the excitation of the color substance (dye) fluorescence happens in a small volume in which the intensity of the excitation is particularly high. With the use of a confocal arrangement, this area is only insignificantly larger than the detected area. The use of a confocal pinhole can therefore be skipped and the detection can happen directly after the objective lens OB as non-descanned detection.
In another arrangement for detection of a dye fluorescence excited through multiphoton absorption, a descanned detection is carried out; however, this time, the pupil of the lens is reproduced in the detection unit (non-confocal descanned detection).
With both the detection arrangements together with the corresponding single-photon or multiphoton absorption only the level (optical section) is reproduced by a three-dimensional lighted picture which lies at the focal plane of the lens. By recording several optical levels in the x-y plane at different depths z of the specimen SP, subsequently a three dimensional picture of the specimen can be generated with the assistance of computers.
The LSM is thus suitable for investigation of thick preparations. The excitation wavelengths are determined by the used dye with its specific absorption properties. The dichroic filters adapted to the emission characteristics of the dye make sure that only fluorescence light emitted by the respective dye is measured by the point detector.
At present, in biomedical applications, several different cell regions are labeled with different dyes at the same time (multifluorescence). The individual dyes can be separately reproduced with the state of the technology on the basis of either different absorption characteristics or emission characteristics (spectra). For this, an additional division of the fluorescence light from several dyes is done with the secondary beam splitters DBS and a separate detection of the individual dye emissions in separate point detectors PMT x.
The LSM LIVE of the Carl Zeiss MicroImaging GmbH realizes a very fast line scanner with a picture creation of about 120 pictures per second http://www.zeiss.de/c12567be00459794/Contents-Frame/fd9fa0090eee01a641256a550036267b.